Small Interfering RNA Specific For HCV And Therapeutic Agent For Hepatitis C Comprising The Same

a technology of hcv and therapeutic agent, applied in the field of small interfering rna specific for hcv, can solve the problems of inability to develop new therapies to treat hcv infection, limited efficacy of current therapeutic regimens, and inability to protect infection with vaccines,

Inactive Publication Date: 2009-12-24
MOGAM BIOTECH RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0019]To increase the stability of synthetic siRNA or the specific interaction between viral target RNA and siRNA Fragment, the 3′-ends of both strands of siRNA were extended with dTdT via chemical synthesis. In a preferred embodiment, synthetic siRNA can be modified internally with chemical derivatives or tagging-molecules for acquiring its physiological stability and chasing its distribution in cells.
[0025]To increase the stability of synthetic siRNA or the specific interaction between target RNA and siRNA fragment, the 3′-ends of both strands of siRNA were extended with dTdT via chemical synthesis. In the siRNA expression vector, two nucleotides of UU at the 3′-ends are generated from polymerase III termination sequences at the final stage of RNA transcription. RNA interference (RNAi) effect is dependent on the detection time and transfected DNA dose and causes lower than 50% and 60% of inhibition of protein expression and viral RNA transcription level, respectively. As the amount of enhanced green fluorescent protein (EGFP) expressed from the siRNA vector presents 50% of the transfection efficiency by FASC analysis, it can be considered that the selected siRNAs induce dramatic and specific anti-viral effect. Especially, the siRNA expression cassette, separated from the vector by PCR amplification with 5′-phosphorylated primers, is the essential element inducing the RNAi effect.

Problems solved by technology

However, there is no vaccine available to protect its infection.
But, current therapeutic regimens have limited efficacy and a poor response rate against certain HCV genotypes.
Therefore, the development of new therapies to treat HCV infection is urgent.
Molecular and immunological studies of HCV replication have been hampered by the lack of a convenient animal model and available in vitro cell culture system.
Although primary infection of the cultured cells with high-titer HCV-patient sera occurred, the condition of efficient viral amplification for detailed studies of HCV replication was not established.

Method used

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  • Small Interfering RNA Specific For HCV And Therapeutic Agent For Hepatitis C Comprising The Same
  • Small Interfering RNA Specific For HCV And Therapeutic Agent For Hepatitis C Comprising The Same
  • Small Interfering RNA Specific For HCV And Therapeutic Agent For Hepatitis C Comprising The Same

Examples

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example 1

siRNA and siRNA Expression Vector

[0052]In mammalian cells, previously siRNA vectors have been designed to transcribe short hairpin RNAs (shRNAs) from an RNA polymerase III promoter (such as U6, H1, and tRNA promoters) or a polymerase II promoter with a poly(A) signal sequence (Brummelkamp et al., Cancer Cell, 2: 243, 2002; Tushcl, Nat. Biotechnol., 20: 446, 2002; Xia et al., Nat. Biotechnol., 20: 1006, 2002). However, shRNA vectors show multiple drawbacks. Their non-natural secondary structure induces that it is hard to synthesize them in bacteria and to sequence, and DNA oligomers to generate them can be costly in the case of high through-put screening. Moreover, it is less facile to generate an siRNA expression cassette containing a promoter to a termination signal without additional sequences for constructing diverse siRNA library. To circumvent these limitations of shRNA expression vectors, the present inventors constructed a vector for direct expression of siRNA, which is trans...

example 2

Inhibition of Protein Expression of FK / R2an Replicon by HCV Specific siRNAs

[0057]The Huh-7 cell line and its replicon cell line that stably supports HCV full-length replicon RNA of genotype 1b (Huh-7FK / R2AN) were obtained from Dr. S. K. Jang (PanBioNet, Korea). The FK / R2AN replicon is a modification from the HCV full-length replicon RNA, FK / RN (Korean Patent Publication No. 2004-32341), with the insertion of the signal sequence of foot-and-mouth disease virus (FMDV) 2A protease, which locates between Rluc and neo coding regions. Cell lines carrying HCV replicons were grown in DMEM containing 10% FBS (Gibco, USA) and 600 mg / ml G418 (Calbiochem, USA) and used in all experiments.

[0058]Transfection of DNAs of the siRNA expression plasmids or PCR products, or synthetic siRNA oligonucleotides was performed in 12-well plate using Lipofectamine 2000 reagent (Invitrogen, USA) in accordance with the manufacturer's instructions. Briefly, 1.5×105 Huh-7 FK / R2AN cells were plated, and the followi...

example 3

Reduction of Viral Transcripts by Vector-Mediated HCV siRNAs

[0067]Total RNA was extracted from FK / R2AN cells transfected with negative control or HCV replicon-specific siRNA expression vectors, at day 2 posttransfection, using Trizol LS reagent (Invitrogen, USA) according to the manufacturer's instruction. The isolated total RNA was digested with RNase-free DNase (Promega, USA). Finally, absolute amount of RNA was quantified by measuring UV-absorbance at 260 nm / 280 nm using UV-spectrophotometer.

[0068]In vitro antiviral activity was assessed by means of a quantitative real-time RT-PCR (Sequence Detection System 5700; Applied Biosystems, USA). The real-time RT-PCR was performed with 500 ng of total RNAs isolated from the transfected cells in a reaction volume of 50 μl using the TaqMan One-Step RT-PCR Master Mix Reagents (Applied Biosystems, USA). Standard RNA was prepared by in vitro transcription of 5′ UTR RNA by T7 RNA polymerase. The primer and probe sequences, specific for HCV 5′U...

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Abstract

The present invention relates to a therapeutic reagent for hepatitis C comprising HCV specific short interfering RNA (siRNA) as an effective ingredient. The siRNA of the invention is a double-stranded RNA specific for the nucleotide sequence of HCV which induces viral RNA degradation in mammalian cells and thereby inhibits HCV protein expression and replication. The method of the invention, which includes the step of administrating the synthetic siRNA or a DNA vector encoding the RNA, is thus effective for the treatment of HCV carrier by inhibiting HCV gene expression and replication.

Description

TECHNICAL FIELD[0001]The present invention relates to a therapeutic agent for hepatitis C comprising HCV specific short interfering RNA (siRNA) as an effective ingredient.BACKGROUND ART[0002]It is estimated that over 270 million people worldwide are chronically infected with Hepatitis C virus (HCV). From 40 to 60% of the patients with this viral pathogen may progress to liver cirrhosis or hepatocellular carcinoma (HCC). However, there is no vaccine available to protect its infection. One of the major anti-HCV therapies is treatment of alpha interferon (IFN-α) alone or in combination with ribavirin. But, current therapeutic regimens have limited efficacy and a poor response rate against certain HCV genotypes. Therefore, the development of new therapies to treat HCV infection is urgent.[0003]RNA interference (RNAi) is evolutionally conserved process in which (endogenous and exogenous) gene expression is suppressed by introduction of double-stranded RNA (dsRNA) in all eukaryotes. RNAi ...

Claims

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Application Information

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IPC IPC(8): A61K48/00C07H21/02C12N15/63C12N15/113
CPCC12N15/1131C12N2310/53C12N2310/14C12N2310/111A61P31/14C12N15/113C12N15/85
Inventor KIM, MEEHYEINSHIN, DUCKHYANGPARK, MAHNHOONKIM, SOO IN
Owner MOGAM BIOTECH RES INST
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